Cytomegalovirus antigens and uses  thereof

ABSTRACT

The disclosure provides modified cytomegalovirus (CMV) gL proteins and complexes comprising gL proteins. The modified gL proteins remain intact and are able to form complexes with other CMV proteins.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 14, 2016, isnamed VN056504WO-SL.txt and is 26,466 bytes in size.

FIELD OF THE INVENTION

This invention is in the field of cytomegalovirus (CMV) antigens thatcan be used for vaccines.

BACKGROUND OF THE INVENTION

Cytomegalovirus is a genus of virus that belongs to the viral familyknown as Herpesviridae or herpesviruses. The species that infects humansis commonly known as human cytomegalovirus (HCMV) or human herpesvirus-5(HHV-5). Within Herpesviridae, HCMV belongs to the Betaherpesvirinaesubfamily, which also includes cytomegaloviruses from other mammals.

Although they may be found throughout the body, HCMV infections arefrequently associated with the salivary glands. HCMV infects between 50%and 80% of adults in the United States (40% worldwide), as indicated bythe presence of antibodies in much of the general population. HCMVinfection is typically unnoticed in healthy people, but can belife-threatening for the immunocompromised, such as HIV-infectedpersons, organ transplant recipients, or new born infants. HCMV is thevirus most frequently transmitted to a developing fetus. Afterinfection, HCMV has an ability to remain latent within the body for thelifetime of the host, with occasional reactivations from latency. Giventhe severity and importance of this disease, obtaining an effectivevaccine is considered a public health top priority (Sung, H., et al.,(2010) Expert review of vaccines 9, 1303-1314; Schleiss, Expert OpinTher Pat. April 2010; 20(4): 597-602).

The genomes of over 20 different HCMV strains have been sequenced,including those of both laboratory strains and clinical isolates. Forexample, the following strains of HCMV have been sequenced: Towne(GL239909366), AD169 (Gl:219879600), Toledo (GL290564358) and Merlin(Gl: 155573956). HCMV strains AD169, Towne and Merlin can be obtainedfrom the American Type Culture Collection (ATCC VR538, ATCC VR977 andATCC VR1590, respectively).

Cytomegalovirus contains an unknown number of membrane proteincomplexes. Of the approximately 30 known glycoproteins in the viralenvelope, gH and gL have emerged as particularly interesting due totheir presence in several different complexes: dimeric gH/gL, trimericgH/gL/gO (also known as the gCIII complex), and the pentamericgH/gL/pUL128/pUL130/pUL131 (pUL131 is also referred to as “pUL131A”,“pUL131a”, or “UL131A”; pUL128, pUL130, and pUL131 subunits sometimesare also referred as UL128, UL130, UL131). CMV is thought to use thepentameric complexes to enter epithelial and endothelial cells byendocytosis and low-pH-dependent fusion but it is thought to enterfibroblasts by direct fusion at the plasma membrane in a processinvolving gH/gL or possibly gH/gL/gO. The gH/gL and/or gH/gL/gOcomplex(es) is/are sufficient for fibroblast infection, whereas thepentameric complex is required to infect endothelial and epithelialcells.

The pentameric complex is considered as a major target for CMVvaccination. Viral genes UL128, UL130 and UL131 are needed forendothelial entry (Hahn, Journal of Virology 2004; 78:10023-33).Fibroblast-adapted non-endothelial tropic strains contain mutations inat least one of these three genes. Towne strain, for example, contains atwo base pair insertion causing a frame shift in UL130 gene, whereasAD169 contains a one base pair insertion in UL131 gene. Both Towne andAD169 could be adapted for growth in endothelial cells, and in bothinstances, the frame shift mutations in UL130 or UL131 genes wererepaired.

U.S. Pat. No. 7,704,510 discloses that pUL131A is required forepithelial cell tropism. U.S. Pat. No. 7,704,510 also discloses thatpUL128 and pUL130 form a complex with gH/gL, which is incorporated intovirions. This complex is required to infect endothelial and epithelialcells but not fibroblasts. Anti-CD46 antibodies were found to inhibitHCMV infection of epithelial cells.

CMV vaccines tested in clinical trials include Towne vaccine,Towne-Toledo chimeras, an alpha virus replicon with gB as the antigen,gB/MF59 vaccine, a gB vaccine produced by GlaxoSmithKline, and a DNAvaccine using gB and pp65. pp65 is viral protein that is a potentinducer of CD8+ responses directed against CMV. These vaccines are allpoor inducers of antibodies that block viral entry intoendothelial/epithelial cells (Adler, S. P. (2013), British MedicalBulletin, 107, 57-68. doi:10.1093/bmb/ldt023).

Preclinical animal studies in CMV vaccines include an inactivated AD169which has been repaired in the UL131 gene, a DNA vaccine using awild-type UL130 gene and peptide vaccines using peptides from pUL130 and131 (Sauer, A, et al., Vaccine 2011; 29:2705-1, doi:10.1016).

CMV gB antigen is considered a poor inducer of antibodies that blockentry into endothelial/epithelial cells. In a Phase II clinical trial,the gB/MF59 vaccine was only 50% effective at preventing primaryinfection among young women with a child at home (Pass, R F, et al., NEngl J Med 2009; 360:1191-9).

Therefore, there is a need for developing CMV vaccines comprising otherantigen targets, such as gH/gL, gH/gL/gO, or pentameric complexgH/gL/pUL128/pUL130/pUL131.

SUMMARY OF THE INVENTION

As disclosed and exemplified herein, the inventors discovered that whenthe cytomegalovirus antigen gL is recombinantly expressed and purifiedfrom a mammalian host (such as a CHO cell or a HEK cell), a significantportion of gL is cleaved. To improve the recombinant expression andpurification of intact gL protein, mutations were introduced to reduceprotease cleavage of gL. The mutants exhibit increased resistance toprotease cleavage during recombination production.

Accordingly, in one aspect, the invention provides a recombinant CMV gLprotein, or a complex-forming fragment thereof, wherein said gL proteinor fragment comprises a mutation at Protease Recognition Site, whereinsaid mutation reduces protease cleavage at said Protease RecognitionSite, as compared to a control. Protease Recognition Site refers toresidues 91-102 (numbering based on SEQ ID NO: 1). Preferably, themutation reduces protease cleavage as compared to a control, withoutchanging the secondary structure of the C-terminal portion of ProteaseRecognition Site (which is believed to have a β-strand conformation).

Also provided herein are CMV complexes comprising the gL proteins orfragments described herein. Such complexes can be gH/gL complex,gH/gL/gO complex, and pentameric complex gH/gL/pUL128/pUL130/pUL131.

Also provided herein are nucleic acids encoding CMV gL proteins andcomplex-forming fragments thereof, as described herein. The nucleic acidmay be used as a nucleic acid-based vaccine (e.g., a self-replicatingRNA molecule encoding the gL or a complex-forming fragment thereof). Thenucleic acid may also be used for recombinant production of gL proteinsor fragments, or a CMV complex comprising the gL proteins or fragments.

The invention also provides a host cell comprising the nucleic acidsdescribed herein. The nucleic acids can be used by the host cell toexpress a gL protein or a complex-forming fragment thereof, or a CMVcomplex comprising the gL or complex-forming fragment thereof.Preferably, the CMV complex can be secreted from the host cell.Preferred host cells are mammalian host cells, such as CHO cells orHEK-293 cells.

The invention also provides a cell culture comprising the host celldescribed herein. Preferably, the culture is at least 20 liters in size.When used for expressing CMV pentameric complexgH/gL/pUL128/pUL130/pUL131, it is preferred that the yield of pentamericcomplex is at least 0.1 g/L.

The invention also provides a method of inducing an immune responseagainst CMV, comprising administering to a subject in need thereof animmunologically effective amount of the gL protein, or a complex-formingfragment thereof, or a CMV complex comprising the gL protein orfragment, as described herein. The invention also provides a method ofinhibiting cytomegalovirus (CMV) entry into a cell, comprisingcontacting the cell with the gL protein, or a complex-forming fragmentthereof, or a CMV complex comprising the gL protein or fragment, asdescribed herein.

Also provided are use of the compositions described herein for inducingan immune response against CMV, and use of the compositions describedherein in the manufacture of a medicament for inducing an immuneresponse against CMV.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the partial sequence alignment of gL proteins fromdifferent herpes viruses near the Protease Recognition Site (SEQ ID NOS12-15, respectively, in order of appearance). FIG. 1B shows thesecondary structure of gH/gL complex from HSV-2 and VZV. The arrowindicates the cleavage site.

FIG. 2A shows the partial sequences of gL mutants (SEQ ID NOS 15-26,respectively, in order of appearance). FIG. 2B shows the result ofwestern blot using anti-gL antibodies. FIG. 2C shows the result ofwestern blot using anti-His antibodies.

FIG. 3 shows western blot analysis of WT and LSG mutant penta usingeither non-reduced (NR) or reduced and boiled (RB) protein samples.

FIG. 4 shows western blot analysis of WT and IDG mutant penta usingeither non-reduced (NR) or reduced and boiled (RB) protein samples.

FIG. 5A shows purified WT penta and IDG and LSG mutant penta. FIG. 5Bshows the neutralization antibody titer (NAB) of mouse serum immunizedwith WT, LSG mutant, or IDG mutant penta adjuvented with MF59.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

As disclosed and exemplified herein, the inventors discovered that whencytomegalovirus antigen gL is recombinantly expressed and purified froma mammalian host (such as a CHO cell or a HEK cell), a significantportion of gL is cleaved (also referred to as “gL clipping”) by anunknown protease. In fact, it was observed that gL clipping occurredduring the recombinant expression and purification of three differentCMV complexes: gH/gL complex, gH/gL/gO complex, and pentameric complexgH/gL/pUL128/pUL130/pUL131. The clipping of gL caused non-homogeneity ofantigen production, and potential loss of neutralizing sites on gL-basedantigens.

Using western blot and N-terminal sequencing, the inventors identifiedand mapped the cleavage site to peptide bond between residues 97 and 98of gL from the Merlin strain (SEQ ID NO:1) (FIGS. 1A-1B). To solve theclipping problem, the inventors studied the structure features of gLproteins from several related herpes viruses, including HSV1, HSV2, andVZV. The gL proteins from HSV1, HSV2, and VZV do not appear to haveclipping problem. Based on the structural studies, the inventorsdiscovered that mutations can be introduced to Protease RecognitionSite, comprising amino acid residues 91 and 102, to reduce the proteasecleavage of recombinantly expressed gL.

For example, as exemplified herein, A96L/N97S/S98G triple mutation (the“LSG” mutant) and A96I/N97D/S98G triple mutation (the “IDG” mutant)substantially eliminated the gL clipping problem. Two other mutants,deletion of residue Asn97 (delta Asp97), and A96S/N97S/S98T (the “SST”mutant), also showed dramatically decreased gL clipping when gH and gLwere co-expressed.

Based on structural analysis of gL proteins from other herpes viruses(FIGS. 1A-1B), it appears that the Protease Recognition Site adopts,from N-terminus to C-terminus, a possible short α-helix (⁹¹VTPE⁹⁴) (SEQID NO: 27), a short loop (⁹⁵AA⁹⁶), and a conserved β-strand structure(⁹⁷NSVLLD¹⁰²) (SEQ ID NO: 7). Cleavage occurs at the N-terminal end ofthe β-strand (FIGS. 1A-1B). A β-strand is a structural unit of β-sheetsin proteins. This is an extended stretch of polypeptide chain typically3 to 10 amino acids long that forms hydrogen bonds with other β-strandsin the same β-sheet. As shown in (FIGS. 1A-1B) this β-strand (β4 inFIGS. 1A-1B together with strands β5 and β6 from gL, as well asβ-strands from gH, form a β-sheet. Therefore, in preferred embodiments,the mutation should maintain the secondary structure of the C-terminalportion of the Protease Recognition Site (i.e., the β-strandconformation is preserved, such as the interactions between β4 and otherβ-strand(s) are substantially maintained). Maintaining the β-strandstructure can potentially reduce any negative impact on the assembly ofCMV complexes (such as pentameric complexes), and can also potentiallypreserve important immunogenic epitopes. For example, one or moreresidues from the Protease Recognition Site can be substituted by acorresponding residue from another herpes virus (such as HSV-1, HSV-2,or VZV). As shown in FIGS. 1A-1B sequence and structural analysis showsthat substituting a CMV residue with a corresponding HSV-1, HSV-2, orVZV residue does not change the β-strand conformation, while proteasecleavage can be reduced. Optionally, the short loop structureimmediately preceding the β-strand (⁹⁵AA⁹⁶ in FIGS. 1A-1B may also bemaintained.

Accordingly, in one aspect, the invention provides a recombinantcytomegalovirus (CMV) gL protein, or a complex-forming fragment thereof,wherein said gL protein or fragment comprises a mutation at ProteaseRecognition Site, wherein said mutation reduces protease cleavage atsaid Protease Recognition Site, as compared to a control. ProteaseRecognition Site refers to residues 91-102 (numbering based on SEQ IDNO:1). Preferably, the mutation reduces protease cleavage as compared toa control, without changing the β-strand structure at the C-terminalportion of the Protease Recognition Site.

Also provided herein are CMV complexes comprising the gL proteins orfragments described herein. Such complexes can be gH/gL complex,gH/gL/gO complex, and pentameric complex gH/gL/pUL128/pUL130/pUL131.

Also provided herein are host cells for recombinant expression of gLproteins or fragments described herein, and CMV complexes comprising gLproteins or fragments described herein. As noted, gL clipping wasobserved in mammalian host cells during the recombinant productionprocess. Therefore, the mutations disclosed herein are particularlysuitable for recombinant production of CMV vaccines in mammalian hosts(which are preferred hosts for many biologics). For example, HEK-293 andCHO cells have long been used for commercial production of biologicalproduction. Therefore, incorporating mutations that reduce gL cleavagecan improve production efficiency and yield, and reduce the formation ofcontaminating, partially degraded product.

2. Definitions

The term “complex-forming fragment” of a cytomegalovirus (CMV) protein(such as gL) refers to any part or portion of the protein that retainthe ability to form a complex with another CMV protein. Such complexesinclude, e.g., gH/gL dimeric complex, gH/gL/gO trimeric complex, orgH/gL/pUL128/pUL130/pUL131 pentameric complex. A “pentamer-formingfragment” of a CMV protein (such as gL) refers to any part or portion ofthe protein that retain the ability to form gH/gL/pUL128/pUL130/pUL131pentameric complex.

As used herein, “pentameric complex” or “pentamer” refers to a CMVcomplex that comprises five different subunits: gH, gL, pUL128, pUL130,and pUL131. Although generally referred to as gH/gL/pUL128/pUL130/pUL131pentamer (or pentameric complex comprising gH, gL, pUL128, pUL130, andpUL131) in the specification, each of the five subunits does not need tobe full-length; the term also encompasses pentamers formed bycomplex-forming fragments of gH, gL, pUL128, pUL130, and pUL131.

The term “mutation” refers to addition, deletion, or substitution of anamino acid residue. The term also includes modifications that introducea non-naturally occurring amino acid or an amino acid analog into apolypeptide chain.

Charged amino acid residues include: D, E, K, R, and H. Polar,non-charged residues include: S, T, C, Y, N, and Q. Nonpolar orhydrophobic residues include: A, V, L, I, M, W, F, and P.

Amino acid residues comprising a large side chain include: W, F, M, Y,Q, R, E, H, and K. Amino acid residues lack of a side chain orcomprising a small side chain include: G, A, V, S, T, C, D, and N.

An amino acid residue comprises a “bulky side chain” when the side chaincomprises a branched or cyclic substituent. Examples of amino acidresidues with a bulky side chain include tryptophan, tyrosine,phenylalanine, homophenylalanine, leucine, isoleucine, histidine,1-methyltryptophan, α-methyltyrosine, α-methylphenylalanine,α-methylleucine, α-methylisoleucine, α-methylhistidine,cyclopentylalanine, cyclohexylalanine, naphthylalanine, etc.

Although the present invention is applicable to gL proteins originatingfrom any CMV strain, in order to facilitate its understanding, whenreferring to amino acid positions in the present specification, thenumbering is given in relation to the amino acid sequence of the gLprotein of SEQ ID NO:1 originating from the Merlin strain, unlessotherwise stated. The present invention is not, however, limited to theMerlin strain. Using the teachings of the present invention, comparableamino acid positions in a gL protein of any other CMV strain can bedetermined by those of ordinary skill in the art by aligning the aminoacid sequences using readily available and well-known alignmentalgorithms (such as BLAST, using default settings; ClustalW2, usingdefault settings; or algorithm disclosed by Corpet, Nucleic AcidsResearch, 1998, 16(22):10881-10890, using default parameters).Accordingly, when referring to a “CMV gL protein”, it is to beunderstood as a CMV gL protein from any strain (in addition to Merlinstrain). The actual number may have to be adjusted for gL proteins fromother strains depending on the actual sequence alignment.

For example, “Protease Recognition Site” is defined as consisting ofamino acid residues 91-102 particularly consisting of residues 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101 and 102. These numbers are inrelation to the amino acid sequence of the gL protein of SEQ ID NO: 1.Protease Recognition Site from gL proteins of other CMV strains, orother gL mutants or variants, or fragments of gL can be ascertainedusing standard sequence alignment programs that align a query sequencewith SEQ ID NO: 1, and identifies residues that match 91-102 of SEQ IDNO: 1.

Specific amino acid residue positions are also numbered according to SEQID NO: 1. For example, “S98” refers to position 98 of SEQ ID NO: 1(which is an S), as well as corresponding residues from other gLsequences (or variants or fragments) that match with S98 of SEQ ID NO:1, when the sequence is aligned with SEQ ID NO: 1. For simplicity, anyresidue from a gL sequence (or variant or fragment) that corresponds toS98 of SEQ ID NO: 1 is referred to as S98, although the actual positionof that residue may or may not be 98, and the actual residue may or maynot be S. For example, a conservative substitution (e.g., T) may bealigned with S98 of SEQ ID NO: 1. A conservative substitution istypically identified as “positive” or “+” by BLAST 2.

Similarly, mutations are also identified according to the numbering ofSEQ ID NO: 1. For example, S98G means that any residue from a gLsequence (or variant or fragment) that corresponds to S98 of SEQ ID NO:1 is mutated to G.

An amino acid residue of a query sequence “corresponds to” a designatedposition of a reference sequence (e.g., S98 of SEQ ID NO: 1) when, byaligning the query amino acid sequence with the reference sequence, theposition of the residue matches the designated position. Such alignmentscan be done by hand or by using well-known sequence alignment programssuch as ClustalW2, or “BLAST 2 Sequences” using default parameters.

An “ ” refers to a sequence that is at least 10 amino acid residueslong, and is at least 50% identical to SEQ ID NO: 5. As shown in FIGS.1A-1B, for wild type gL from Merlin strain, a 17-residue fragment uniqueto CMV gL, as compared to HSV1, HSV2, and VZV, has been identified(shown as “1-11”). Preferably, the Insert Region comprises at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 residues, and/or isat least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:5. In certain embodiments, the Insert Region comprises a sequence inwhich one to eight amino acid residues of SEQ ID NO: 5 areconservatively substituted.

“Conservatively substituted” means that a residue is replaced byanother, biologically similar residue. Examples include substitution ofamino acid residues with similar characteristics, e.g. small aminoacids, acidic amino acids, polar amino acids, basic amino acids,hydrophobic amino acids and aromatic amino acids. An example ofconservative amino acid substitutions includes those in the followingTable 1, and analogous substitutions of the original residue bynon-natural alpha amino acids which have similar characteristics.

TABLE 1 Very Highly- Highly Conserved Original Conserved Substitutions(from the Conserved Substitutions Residue Substitutions Blosum90 Matrix)(from the Blosum65 Matrix) Ala Ser Gly, Ser, Thr Cys, Gly, Ser, Thr, ValArg Lys Gln, His, Lys Asn, Gln, Glu, His, Lys Asn Gln; His Asp, Gln,His, Lys, Ser, Thr Arg, Asp, Gln, Glu, His, Lys, Ser, Thr Asp Glu Asn,Glu Asn, Gln, Glu, Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, His, Lys,Met Arg, Asn, Asp, Glu, His, Lys, Met, Ser Glu Asp Asp, Gln, Lys Arg,Asn, Asp, Gln, His, Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn,Gln, Tyr Arg, Asn, Gln, Glu, Tyr Ile Leu; Val Leu, Met, Val Leu, Met,Phe, Val Leu Ile; Val Ile, Met, Phe, Val Ile, Met, Phe, Val Lys Arg;Gln; Glu Arg, Asn, Gln, Glu Arg, Asn, Gln, Glu, Ser, Met Leu; Ile Gln,Ile, Leu, Val Gln, Ile, Leu, Phe, Val Phe Met; Leu; Tyr Leu, Trp, TyrIle, Leu, Met, Trp, Tyr Ser Thr Ala, Asn, Thr Ala, Asn, Asp, Gln, Glu,Gly, Lys, Thr Thr Ser Ala, Asn, Ser Ala, Asn, Ser, Val Trp Tyr Phe, TyrPhe, Tyr Tyr Trp; Phe His, Phe, Trp His, Phe, Trp Val Ile; Leu Ile, Leu,Met Ala, Ile, Leu, Met, Thr

Unless otherwise specified, the percent identity of two sequences isdetermined over the entire length of the shorter of the two sequences.

3. Modified CMV gL Proteins and Complexes

A. Modified gL Proteins

In one aspect, the invention provides a modified CMV gL protein, or acomplex-forming fragment thereof, that reduces clipping (cleavage) atthe peptide bond between residues N97 and S98.

Human CMV glycoprotein L (gL) is encoded by the UL115 gene. gL isthought to be essential for viral replication and all known functionalproperties of gL are directly associated with its dimerization with gH.The gH/gL complex is required for the fusion of viral and plasmamembranes leading to virus entry into the host cell. gL from HCMV strainMerlin (Gl:39842115, SEQ ID NO: 1) and HCMV strain Towne (Gl:239909463,SEQ ID NO: 2) have been reported to be 278 amino acids in length. gLfrom HCMV strain AD169 (Gl:2506510, SEQ ID NO: 3) has been reported tobe 278 amino acids in length, include a signal sequence at itsN-terminus (amino acid residues 1-35), have two N-glycosylation sites(at residues 74 and 114) and lack a TM domain (Rigoutsos, I, et al.,Journal of Virology 77 (2003): 4326-44). The N-terminal signal sequencein SEQ ID NO: 1 is predicted to comprise amino acid residues 1-30. SEQID NO: 2 shares 98% amino acid identity with SEQ ID NO: 1. Sequencing ofthe full-length gL gene from 22 to 39 clinical isolates, as well aslaboratory strains AD169, Towne and Toledo revealed less than 2%variation in the amino acid sequences among the isolates (Rasmussen, L,et al., Journal of Virology 76 (2002): 10841-10888).

Typically, the N-terminal signal sequence of gL proteins is cleaved by ahost cell signal peptidase to produce mature gL proteins. The gLproteins in HCMV membrane complexes of the invention may lack anN-terminal signal sequences. An example of gL protein lacking N-terminalsignal sequences is SEQ ID NO: 4, which lacks an N-terminal signalsequence and consists of amino acid residues 31-278 of SEQ ID NO: 1.

While gL is thought to be essential for viral replication, all knownfunctional properties of gL are directly associated with itsdimerization with gH.

gL proteins of the invention can be gL variants that have variousdegrees of identity to SEQ ID NO: 1 such as at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thesequence recited in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. gLproteins of the invention can have various degrees of identity to SEQ IDNO: 4 such as at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the sequence recited in SEQ ID NO: 4.In certain embodiments, the gL variant proteins: (i) form part of thedimeric complex gH/gL; (ii) form part of the trimeric gH/gL/gO complex;(iii) form part of the pentameric gH/gL/pUL128/pUL130/pUL131 complex;(iv) comprise at least one epitope from SEQ ID NO: 1, SEQ ID NO: 2 , SEQID NO: 3, or SEQ ID NO: 4; and/or (v) can elicit antibodies in vivowhich immunologically cross react with a CMV virion.

Also encompassed in the invention are complex-forming fragments of gLproteins described herein. A complex-forming fragment of gL can be anypart or portion of the gL protein that retains the ability to form acomplex with another CMV protein. In certain embodiments, acomplex-forming fragment of gL forms part of the dimeric complex gH/gL.In certain embodiments, a complex-forming fragment of gL forms part ofthe trimeric gH/gL/gO complex. In certain embodiments, a complex-formingfragment of gL forms part of the pentameric gH/gL/pUL128/pUL130/pUL131complex. A complex-forming fragment of gL can be obtained or determinedby standard assays known in the art, such as co-immunoprecipitationassay, cross-linking, or co-localization by fluorescent staining, etc.In certain embodiments, the complex-forming fragment of gL also (i)comprises at least one epitope from SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, or SEQ ID NO: 4; and/or (ii) can elicit antibodies in vivo whichimmunologically cross react with a CMV virion.

In certain embodiments, the gL protein described herein, or acomplex-forming fragment thereof, comprises a mutation at ProteaseRecognition Site (residues 91-102), wherein said mutation reducesprotease cleavage at said Protease Recognition Site, as compared to acontrol.

A variety of controls may be used. The level of protease cleavage (atpeptide bond between residues 97 and 98) of a corresponding wild type gLunder substantially the same condition can be used as a control.Alternatively, a control may be a pre-determined level or a thresholdlevel (e.g., 20%, 25%, or 30% of the total gL protein). The percentagerefers to molar percentage.

For example, the mutation can result in a reduction in protease cleavageat the peptide bond between residues 97 and 98 by at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% etc., as compared to that of wild type, whenrecombinantly expressed in a mammalian host cell under a standardculturing condition for that host cell.

Alternatively or in addition, the protease cleavage is reduced by atleast 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, atleast 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, atleast 70 fold, at least 75 fold, at least 80 fold, at least 90 fold orat least 100 fold, as compared to that of wild type, when recombinantlyexpressed in a mammalian host cell under a standard culturing conditionfor that host cell.

Alternatively or in addition, the mutation can be one wherein no morethan about 35% of the gL molecules, or complex-forming fragment thereof,are cleaved at a peptide bond between residues 97 and 98, whenrecombinantly expressed in a mammalian host cell under a standardculturing condition for that host cell. For example, the mutation canresult in no more than about 35%, no more than about 30%, no more thanabout 25%, no more than about 20%, no more than about 15%, no more thanabout 10%, no more than about 9%, no more than about 8%, no more thanabout 7%, no more than about 6%, no more than about 5%, no more thanabout 4%, no more than about 3%, no more than about 2%, or no more thanabout 1% of the gL molecules, or complex-forming fragment thereof, arecleaved at peptide bond between residues 97 and 98, when recombinantlyexpressed in a mammalian host cell under a standard culturing conditionfor that host cell. The percentage refers to molar percentage.

Standard culturing conditions for commonly used mammalian host cells areknown. For example, for a CHO cell, a standard culturing condition canbe temperature at 36.5° C. in a pH 7.0 medium, with ≤10% CO₂. In onespecific example, Expi293 cells were transfected to express pentamericcomplex (gH/gL/pUL128/pUL130/pUL131) at 37° C. and in pH 7.0 under 8%CO₂ for three days, and supernatants of the cell culture were affinitypurified and analyzed with western blots, as shown in examples below.

The mutation comprises addition, deletion, substitution, ora combinationthereof, of an amino acid residue. Preferably, the mutationsubstantially preserves the secondary structure of the C-terminalportion of the Protease Recognition Site. In particular, as shown inFIGS. 1A-1B, residues of the C-terminal portion of said ProteaseRecognition Site form a β-strand, which is believed to interact withother β-strands to form a β-sheet. Preferably, said mutation maintainsthis β-strand conformation. Potential advantages of maintaining thesecondary structure include, e.g., facilitating the assembly ofgL-containing complexes (e.g., gH/gL, gH/gL/gO, orgH/gL/pIL128/pUL130/pUL131), and maintaining key immunogenic epitopes.Optionally, the short loop structure immediately preceding the β-strandis also preserved.

Many computer programs and algorithms are available to predict secondarystructure, including I-TASSER, HHpred, RaptorX, MODELLER, SWISS-MODEL,Robetta Beta, SPARKSx, PEP-FOLD, Phyre and Phyre2, RAPTOR, QUARK,Abalone, Foldit, etc. Whether a mutation changes the secondary structureof the Protease Recognition Site can be analyzed using these tools.

In certain embodiments, the mutation comprises addition of one or moreamino acid residues. For example, the mutation can comprise addition oftwo to five amino acid residues. In certain embodiments, the two to fiveamino acid residues comprise both polar residue(s) and non-polarresidue(s).

In certain embodiments, the mutation comprises the addition of one ormore residues between residues N97 and S98. As shown in the Examples,the peptide bond between N97 and S98 is cleaved by a protease;therefore, introducing one or more additional residues between N97 andS98 can result in a mutant gL (or fragment) that is more cleavageresistant. In an exemplary embodiment, the mutation comprises additionof F, Q, or a combination thereof, between residues 97 and 98. In anexemplary embodiment, the mutation comprises addition of FQ or QFbetween residues 97 and 98.

In certain embodiments, the mutation comprises deletion of one or moreamino acid residues, such as deletion of one to three amino acidresidues. In certain embodiments, the mutation comprises the deletion ofat least one residue selected from the group consisting of: V91, T92,P93, E94, A95, A96, N97, S98, V99, L100, L101, D102, and a combinationthereof. In certain embodiments, the mutation comprises deletion of atleast one residue selected from the group consisting of: E94, A95, A96,N97, S98, V99, L100, L101, D102 and a combination thereof. In anexemplary embodiment, the mutation comprises deletion of at least oneresidue selected from the group consisting of: A96, N97, S98, and acombination thereof. In an exemplary embodiment, the mutation comprisesdeleting N97.

In certain embodiments, the mutation comprises substituting a residuewith a corresponding residue from the gL protein of another herpesvirus. Herpes virus (Herpesviridae) family include, e.g., herpes simplexviruses 1 and 2 (HSV-1 or HHV-1, HSV-2 or HHV-2), varicella-zoster virus(VZV or HHV-3), Epstein-Barr virus (EBV or HHV-4), human herpesvirus 6(HHV-6), human herpesvirus 7 (HHV-7), and Kaposi's sarcoma-associatedherpesvirus (HHV-8). In certain embodiments, the gL protein from anotherherpes virus is the gL protein from HSV1, HSV2, VZV, EBV, PrV, or bovineherpesvirus 5.

One potential advantage of substituting a CMV residue with acorresponding residue from another herpes virus is that the secondarystructure of the Protease Recognition Site will likely be preserved. Asshown in FIGS. 1A-1B, HSV-1, HSV-2 and VZV all share substantially thesame secondary structure, especially, the C-terminal portion of theProtease Recognition Sites all adopt a β-stand structure.

If multiple substitutions are made, they do not have to come from thesame herpes virus. For example, one may substitute a first CMV residuewith the corresponding residue from HSV-1, a second residue with thecorresponding residue from HSV-2, and/or a third CMV residue with thecorresponding residue from VZV, etc. Therefore, the mutation maycomprises a first amino acid residue substituted with a correspondingresidue from a first other herpes virus gL protein, and a second aminoacid residue substituted with a corresponding residue from a secondother herpes virus gL protein, and/or a third amino acid residuesubstituted with a corresponding residue from a third other herpes virusgL protein, etc.

In certain embodiments, the mutation comprises substituting E94 with A.

In certain embodiments, the mutation comprises substituting A95 with R,L, or N.

In certain embodiments, the mutation comprises substituting A96 with anon-polar residue or with a residue that comprises a large side chain,such as W, F, or M. In certain embodiments, the mutation comprisessubstituting A96 with I, L, or S.

In certain embodiments, the mutation comprises substituting N97 with apolar residue or a non-polar residue. The polar residue can comprise asmall side chain or a large side chain. In certain embodiments, themutation comprises substituting N97 with S, D, E, A, or Y.

In certain embodiments, the mutation comprises substituting S98 with anamino acid residue with a small side chain, such as G, A, V, S, T, C, D,or N. In certain embodiments, the mutation comprises substituting S98with G, T, V, or I.

In certain embodiments, the mutation comprises substituting V99 with anamino acid residue with I.

In certain embodiments, the mutation comprises substituting L100 with anamino acid residue with F or V.

In certain embodiments, the mutation comprises substituting L101 with anamino acid residue with V.

The addition, deletion, and substitutions described herein can be usedin singular, or in any combination. For example, the gL mutant maycomprise an addition at one position, a deletion at a second position,and a substitution at a third position.

In certain embodiments, the gL protein or fragment comprises an InsertRegion at the N-terminus of the Protease Recognition Site. As shown inFIGS. 1A-1B, as compared to gL proteins from HSV-1, HSV-2, and ZVZ, theCMV gL protein comprises an extra 17-residue insert. As shown in theExamples, when this 17-residue insert was partially or fully deleted,the gL protein became more prone to protease cleavage. Therefore, the17-residue insert appears to at least partially block the access of theprotease to the Protease Recognition Site. Therefore, maintaining anInsert Region at the N-terminus of the Protease Recognition Site may bedesirable. An “Insert Region” should be at least 10 amino acid residueslong, and is at least 50% identical to SEQ ID NO: 5 (which is theoriginal 17-residue fragment unique to CMV gL, as compared to HSV1,HSV2, and VZV).

In certain embodiments, the mutation comprises introducing anon-naturally occurring amino acid residue, which is believed to reducethe protease cleavage.

In certain embodiments, the mutation comprises introducing an amino acidresidue comprising a bulky side chain, which is believed to at leastpartially block the access of the protease to the Protease RecognitionSite, and reduces protease cleavage.

B. CMV Protein Complexes

In another aspect, the invention provides a complex comprising themodified CMV gL protein, or a complex-forming fragment thereof,described herein. Such complexes include, e.g., (i) isolated dimericcomplexes comprising: the modified gL protein, or a complex-formingfragment thereof, described herein, and CMV proteins gH or acomplex-forming fragment thereof; (ii) isolated trimeric complexcomprising the modified gL protein, or a complex-forming fragmentthereof, described herein, and CMV proteins gH or a complex-formingfragment thereof, and gO or a complex-forming fragment thereof; and(iii) isolated pentameric complexes comprising the modified gL protein,or a complex-forming fragment thereof, described herein, and CMVproteins pUL128 or a complex-forming fragment thereof, pUL130 or acomplex-forming fragment thereof, pUL131 or a complex-forming fragmentthereof, and gH or a complex-forming fragment thereof. Also included areany other complexes comprising gL (or a complex-forming fragmentthereof) as a component.

Although gH, gL, gO, pUL128, pUL130, pUL131 may be referred to asglycoproteins, this nomenclature should not be taken to mean that theseproteins must be glycosylated when used with the invention. On thecontrary, in some embodiments of the invention, one or more ofpolypeptides are not glycosylated. Usually, however, one or more (orall) polypeptides in a complex of the invention are glycosylated. Insome embodiments, one or more (or all) polypeptides in a complex of theinvention are glycosylated by glycosylation mutants of cultured cells,such as mutated mammalian cells. Such glycosylation mutants produce apattern of polypeptide glycosylation which differs from a wild-typepattern of glycosylation, i.e. the resulting polypeptide glycoformsdiffer from wild-type glycoforms.

In certain embodiments, the glycosylation pattern of the gL (or acomplex-forming fragment thereof), or a complex comprising gL (or acomplex-forming fragment thereof) has a mammalian glycosylation pattern;and/or does not have an insect cell pattern of glycosylation. In someembodiments, one or more of the proteins of the complex contain complexN-linked side chains with a penultimate galactose and terminal sialicacid.

For recombinant production of protein complexes (such as pentamericcomplex), it may be desirable that the complex is soluble. Based onsequence analysis, CMV gH protein comprises a transmembrane (TM) domain,but gL, gO, pUL128, pUL130, and pUL131 do not have transmembranedomains. So, to produce a soluble complex (e.g., pentameric complex),the gH subunit of the pentameric complex may lack the TM domain. Forexample, a gH fragment comprising the N-terminal signal sequence and theectodomain, but not the TM domain, of gH can be used.

The gH from CMV strain Towne is shown as SEQ ID NO: 6 (Gl:138314, 742amino acid residues). gH from Towne has been characterized as having:(i) six N-glycosylation sites (at residues 55, 62, 67, 192, 641 and700); (ii) a hydrophobic signal sequence at its N-terminus (amino acidresidues 1-23); (iii) an ectodomain (residues 24-717) that projects outof the cell into the extracellular space; (iv) a hydrophobictransmembrane (TM) domain (residues 718-736); and (v) a C-terminalcytoplasmic domain (residues 737-742). The TM domains of gH proteinsfrom other strains, or of other gH variants and fragments, can beidentified according to sequence alignment.

For ease of production, the recombinantly produced CMV complex (such aspentameric complex) may be secreted from the host cell into culturingmedium.

In certain embodiments, said pentameric complex is secreted from thehost cell. It has been reported that the presence of all five subunits,gH, gL, pUL128, pUL131, and pUL131, is sufficient for the assembly ofthe pentameric complex in ER before it is exported to the Golgiapparatus. See, Ryckman et al., J Virol. January 2008; 82(1): 60-70.Alternatively or in addition, an appropriate signal peptide may be usedin one or more of the five subunits (e.g., by making a fusion proteinwith a secretory signal). Signal sequences (and expression cassette) forproducing secretory proteins are known in the prior art. In general,leader peptides are 5-30 amino acids long, and are typically present atthe N-terminus of a newly synthesized protein. The core of the signalpeptide generally contains a long stretch of hydrophobic amino acidsthat has a tendency to form a single alpha-helix. In addition, manysignal peptides begin with a short positively charged stretch of aminoacids, which may help to enforce proper topology of the polypeptideduring translocation. At the end of the signal peptide there istypically a stretch of amino acids that is recognized and cleaved bysignal peptidase. Signal peptidase may cleave either during or aftercompletion of translocation to generate a free signal peptide and amature protein.

C. Nucleic Acid Encoding Modified gL Proteins and Complexes

In another aspect, the invention provides a nucleic acid comprising asequence that encodes the modified gL protein, or a complex-formingfragment thereof, described herein. The nucleic acid can be DNA or RNA.

In certain embodiments, the nucleic acid is DNA. DNA-based expressionsystems for expression and purification of recombinant proteins arewell-known in the art. For example, the expression system may be avector comprising a nucleotide sequence that encodes the modified gL orgL fragment described herein, which is operably linked to an expressioncontrol sequence that regulates the expression of the modified gL or gLfragment in a host cell, such as a mammalian host cell, a bacterial hostcell, or an insect host cell. The expression control sequence may be apromoter, an enhancer, a ribosome entry site, or a polyadenylationsequence, for example. Other expression control sequences contemplatedfor use in the invention include introns and 3′ UTR sequences.

The recombinantly expressed modified gL protein of fragment thereof, ora complex comprising the modified gL protein or fragment thereof can bepurified using methods described herein, such as purification methodsdisclosed in WO 2014/005959, or other methods known in the art.

In certain embodiments, the nucleic acid molecule is a vector derivedfrom an adenovirus, an adeno-associated virus, a lentivirus, or analphavirus. In certain embodiments, the nucleic acid molecule is areplication-deficient viral vector.

In certain embodiments, the nucleic acid is RNA. In certain embodiments,the nucleic acid is a self-replicating RNA molecule, such as analphavirus-derived RNA replicon.

Self-replicating RNA molecules are well known in the art and can beproduced by using replication elements derived from, e.g., alphaviruses,and substituting the structural viral proteins with a nucleotidesequence encoding a protein of interest. A self-replicating RNA moleculeis typically a plus-strand molecule which can be directly translatedafter delivery to a cell, and this translation provides a RNA-dependentRNA polymerase which then produces both antisense and sense transcriptsfrom the delivered RNA. Thus the delivered RNA leads to the productionof multiple daughter RNAs. These daughter RNAs, as well as collinearsubgenomic transcripts, may be translated themselves to provide in situexpression of an encoded antigen, or may be transcribed to providefurther transcripts with the same sense as the delivered RNA which aretranslated to provide in situ expression of the antigen. The overallresult of this sequence of transcriptions is a huge amplification in thenumber of the introduced replicon RNAs and so the encoded antigenbecomes a major polypeptide product of the cells. Cells transfected withself-replicating RNA briefly produce antigen before undergoing apoptoticdeath. This death is a likely result of requisite double-stranded (ds)RNA intermediates, which also have been shown to super-activatedendritic cells. Thus, the enhanced immunogenicity of self-replicatingRNA may be a result of the production of pro-inflammatory dsRNA, whichmimics an RNA-virus infection of host cells.

One suitable system for achieving self-replication in this manner is touse an alphavirus-based replicon. Alphaviruses comprise a set ofgenetically, structurally, and serologically related arthropod-borneviruses of the Togaviridae family. Twenty-six known viruses and virussubtypes have been classified within the alphavirus genus, including,Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelanequine encephalitis virus. As such, the self-replicating RNA of theinvention may incorporate a RNA replicase derived from semliki forestvirus (SFV), sindbis virus (SIN), Venezuelan equine encephalitis virus(VEE), Ross-River virus (RRV), eastern equine encephalitis virus, orother viruses belonging to the alphavirus family.

An alphavirus-based “replicon” expression vectors can be used in theinvention. Replicon vectors may be utilized in several formats,including DNA, RNA, and recombinant replicon particles. Such repliconvectors have been derived from alphaviruses that include, for example,Sindbis virus (Xiong et al. (1989) Science 243:1188-1191; Dubensky etal., (1996) J. Virol. 70:508-519; Hariharan et al. (1998) J. Virol.72:950-958; Polo et al. (1999) PNAS 96:4598-4603), Semliki Forest virus(Liljestrom (1991) Bio/Technology 9:1356-1361; Berglund et al. (1998)Nat. Biotech. 16:562-565), and Venezuelan equine encephalitis virus(Pushko et al. (1997) Virology 239:389-401). Alphaviruses-derivedreplicons are generally quite similar in overall characteristics (e.g.,structure, replication), individual alphaviruses may exhibit someparticular property (e.g., receptor binding, interferon sensitivity, anddisease profile) that is unique. Therefore, chimeric alphavirusreplicons made from divergent virus families may also be useful.

In some embodiments, CMV gL proteins (or fragments thereof) describedherein are delivered using alphavirus replicon particles (VRP). An“alphavirus replicon particle” (VRP) or “replicon particle” is analphavirus replicon packaged with alphavirus structural proteins.

Uses of alphavirus-based RNA replicon are known in the art, see, e.g.,WO 2013006837, paragraphs [0155] to [0179]. The RNA replicon can beadministered without the need for purification of the protein encodedtherein.

In certain embodiments, the nucleic acid molecule is part of a vectorderived from an adenovirus. The adenovirus genome is a lineardouble-stranded DNA molecule of approximately 36,000 base pairs with the55-kDa terminal protein covalently bound to the 5′ terminus of eachstrand. Adenoviral (“Ad”) DNA contains identical Inverted TerminalRepeats (“ITRs”) of about 100 base pairs with the exact length dependingon the serotype. The viral origins of replication are located within theITRs exactly at the genome ends. Adenoviral vectors for use with thepresent invention may be derived from any of the various adenoviralserotypes, including, without limitation, any of the over 40 serotypestrains of adenovirus, such as serotypes 2, 5, 12, 40, and 41.

In certain embodiments, the nucleic acid molecule is part of a vectorderived from an Adeno Associated Virus (AAV). The AAV genome is a linearsingle-stranded DNA molecule containing approximately 4681 nucleotides.The AAV genome generally comprises an internal nonrepeating genomeflanked on each end by inverted terminal repeats (ITRs). The ITRs areapproximately 145 base pairs (bp) in length. The ITRs have multiplefunctions, including serving as origins of DNA replication and aspackaging signals for the viral genome. AAV is a helper-dependent virus;that is, it requires co-infection with a helper virus (e.g., adenovirus,herpesvirus or vaccinia) in order to form AAV virions in the wild. Inthe absence of co-infection with a helper virus, AAV establishes alatent state in which the viral genome inserts into a host cellchromosome, but infectious virions are not produced. Subsequentinfection by a helper virus rescues the integrated genome, allowing itto replicate and package its genome into infectious AAV virions. WhileAAV can infect cells from different species, the helper virus must be ofthe same species as the host cell. Thus, for example, human AAV willreplicate in canine cells co-infected with a canine adenovirus.

In certain embodiments, the nucleic acid molecule is part of a vectorderived from a retroviruses. A selected gene can be inserted into avector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems have been described. See, e.g., U.S. Pat. No. 5,219,740; Millerand Rosman (1989) BioTechniques 7:980-90; Miller, A. D. (1990) HumanGene Therapy 1 :5-14; Scarpa et al. (1991) Virology 180:849-52; Burns etal. (1993) Proc. Natl. Acad. Sci. USA 90:8033-37; Boris-Lawrie and Temin(1993) Curr. Opin. Genet. Develop. 3 : 102-09.

The invention also provides host cells comprising the nucleic acidmolecules disclosed herein. Host cells suitable for harboring thenucleic acid molecules and/or for expressing recombinant proteins, andmethods of introducing a nucleic acid into a suitable host cell, areknown in the art.

4. Recombinant Production of gL Proteins and Complexes

The invention also provides a host cell comprising the nucleic acidsencoding the gL protein and fragment thereof, as described above.

Preferably, the host cells are mammalian cells (e.g., human, non-humanprimate, horse, cow, sheep, dog, cat, and rodent (e.g., hamster), aviancells (e.g., chicken, duck, and geese). Suitable mammalian cellsinclude, for example, Chinese hamster ovary (CHO) cells, human embryonickidney cells (HEK-293 cells, typically transformed by sheared adenovirustype 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, HeLa cells, PERC.6cells (ECACC deposit number 96022940), Hep G2 cells, MRC-5 (ATCCCCL-171), WI-38 (ATCC CCL-75), fetal rhesus lung cells (ATCC CL-160),Madin-Darby bovine kidney (“MDBK”) cells, Madin-Darby canine kidney(“MDCK”) cells (e.g., MDCK (NBL2), ATCC CCL34; or MDCK 33016, DSM ACC2219), baby hamster kidney (BHK) cells, such as BHK21-F, HKCC cells, andthe like.

In certain embodiments, the host cell is a HEK-293 cell. In certainembodiments, the host cell is a CHO cell. In certain embodiments, thepolynucleotide encoding the gL protein (or fragment thereof) describedherein is integrated into the genomic DNA of the CHO cell. Forrecombinant production of a CMV protein complex, the nucleotide sequenceencoding other subunits of the complex should also be integrated intothe genomic DNA of the CHO cell.

Accordingly, in certain embodiments, the host cell comprises one or morepolynucleotide sequences encoding CMV pentameric complex, saidpentameric complex comprising: gH or a pentamer-forming fragmentthereof, gL or a pentamer-forming fragment thereof, pUL128 or apentamer-forming fragment thereof, pUL130 or a pentamer-forming fragmentthereof, and pUL131 or a pentamer-forming fragment thereof. In certainembodiments, the one or more polynucleotide sequences encoding CMVpentameric complex are integrated into the genomic DNA of said hostcell. In certain embodiments, the host cell, when cultured under asuitable condition, expresses said CMV pentameric complex (which ispreferably soluble and/or secreted from the host cell).

Exemplary CHO cell lines available at European Collection of CellCultures (ECACC) are listed in Table 2. Any CHO cells listed in Table 2may be used.

TABLE 2 Cell Line Name Keywords CHO Hamster Chinese ovary CHO (PROTEINFREE) Chinese hamster ovary CHO-CHRM1 Human cholinergic receptormuscarinic M1, CHRM1, G Protein Coupled Receptor, GPCR, Transfected,InSCREENeX SCREENflex ™, CHO-K1 Host. CHO-CHRM2 Human cholinergicreceptor muscarinic M2, CHRM2, G Protein Coupled Receptor, GPCR,Transfected, InSCREENeX SCREENflex ™, CHO-K1 Host. CHO-CHRM5 Humancholinergic receptor muscarinic M5, CHRM5, G Protein Coupled Receptor,GPCR, Transfected, InSCREENeX SCREENflex ™, CHO-K1 Host. CHO-CNR1 Humancannabinoid receptor I, CNR1 Gene ID 1268, G Protein Coupled Receptor,GPCR, Transfected, InSCREENeX SCREENflex ™, CHO-K1 Host. CHO-FFAR2 Humanfree fatty acid receptor 2, FFAR2, G Protein Coupled Receptor, GPCR,Transfected, InSCREENeX SCREENflex ™, CHO-K1 Host. CHO-GPR120 Humanreceptor GPR120 (orphan), GPR120, G Protein Coupled Receptor, GPCR,Transfected, InSCREENeX SCREENflex ™, CHO-K1 Host. CHO-K1 HamsterChinese ovary CHO-K1-AC-free Hamster Chinese Ovary, serum-free CHO-K1/SFHamster Chinese ovary (MEM adapted) CHO-NPY1R Human neuropeptide Yreceptor, NPY1R, Gene ID 4886, G Protein Coupled Receptor, GPCR,Transfected, InSCREENeX SCREENflex ™, CHO-K1 Host. CHO-OPRL1 Humanopiate receptor-like 1, OPRL1, G Protein Coupled Receptor, GPCR,Transfected, InSCREENeX SCREENflex ™, CHO-K1 Host. CHO-SSTR1 HumanSomatostatin Receptor 1, SSTR1 G Protein Coupled Receptor, GPCR,Transfected, InSCREENeX SCREENflex ™, CHO-K1 Host. CHO/dhFr- HamsterChinese ovary CHO/dhFr-AC-free Hamster Chinese Ovary, serum-freeRR-CHOKI Hamster Chinese ovary T02J-10/10 (CHO-GCGR Human glucagonreceptor, GCGR, G Protein Coupled Receptor, GPCR, (GCGR)) Transfected,InSCREENeX SCREENflex ™, CHO-K1 Host.

Various CHO cell lines are also available from American Type CultureCollection (ATCC), such as CHO cell lines hCBE11 (ATCC® PTA-3357™),E77.4 (ATCC® PTA-3765™), hLT-B: R-hG1 CHO #14 (ATCC® CRL-11965™),MOR-CHO-MORAb-003-RCB (ATCC® PTA-7552™), AQ.C2 clone 11B (ATCC®PTA-3274™), AQ.C2 clone 11B (ATCC® PTA-3274™), hsAQC2 in CHO-DG44 (ATCC®PTA-3356™), xrs5 (ATCC® CRL-2348™), CHO-K1 (ATCC® CCL-61™), Lec1[originally named Pro-5WgaRI3C] (ATCC® CRL-1735™), Pro-5 (ATCC®CRL-1781™), ACY1-E (ATCC® 65421™), ACY1-E (ATCC® 65420™), pgsE-606(ATCC® CRL-2246™), CHO-CD36 (ATCC® CRL-2092™), pgsC-605 (ATCC®CRL-2245™), MC2/3 (ATCC® CRL-2143™), CHO-ICAM-1 (ATCC® CRL-2093™), andpgsB-618 (ATCC® CRL-2241™). Any one of these CHO cell lines may be used.

Other commercially available CHO cell lines include, e.g., FreeStyle™CHO-S Cells and Flp-In™-CHO Cell Line from Life Technologies.

Other suitable host cells include, e.g., a CHO cell in which theexpression level or activity of C12orf35 protein is reduced, as comparedto a control (see, e.g., WO2015/092735, incorporated herein byreference, which provides a detailed description of mammalian cellswherein the expression level or activity of C12orf35 protein is reducedas compared to a control), a CHO cell in which the expression level oractivity of FAM60A protein is reduced, as compared to a control (see,e.g., WO2015/092737, incorporated herein by reference, which provides adetailed description of mammalian cells wherein the expression level oractivity of FAM60A protein is reduced); a CHO cell in which theexpression level or activity of matriptase is reduced, as compared to acontrol (U.S. Provisional Patent application No. 61/985,589, filed Apr.29, 2014 and incorporated herein by reference, and U.S. ProvisionalPatent Application No. 61/994,310, filed May 16, 2014 and incorporatedherein by reference, provides a detailed description of mammalian cellswherein the expression level or activity of matriptase is reduced).

Methods for expressing recombinant proteins in CHO cells in general havebeen disclosed. See, e.g., in U.S. Pat. Nos. 4,816,567 and 5,981,214.

EP patent application EP14191385.5 filed Oct. 31, 2014 (incorporatedherein by reference) discloses mammalian host cells, in particular CHOcells, in which the sequence(s) encoding CMV proteins gH, gL, pUL128,pUL130, pUL131 (or a complex-forming fragment thereof) are stablyintegrated into the genome.

Also provided herein is a cell culture comprising the host celldescribed herein. The cell culture can be large scale, e.g., at leastabout 10 L, at least about 20 L, at least about 30 L, at least about 40L, at least about 50 L, at least about 60 L, at least about 70 L, atleast about 80 L, at least about 90 L, at least about 100 L, at leastabout 150 L, at least about 200 L, at least about 250 L, at least about300 L, at least about 400 L, at least about 500 L, at least about 600 L,at least about 700 L, at least about 800 L, at least about 900 L, atleast about 1000 L, at least about 2000 L, at least about 3000 L, atleast about 4000 L, at least about 5000 L, at least about 6000 L, atleast about 10,000 L, at least about 15,000 L, at least about 20,000 L,at least about 25,000 L, at least about 30,000 L, at least about 35,000L, at least about 40,000 L, at least about 45,000 L, at least about50,000 L, at least about 55,000 L, at least about 60,000 L, at leastabout 65,000 L, at least about 70,000 L, at least about 75,000 L, atleast about 80,000 L, at least about 85,000 L, at least about 90,000 L,at least about 95,000 L, at least about 100,000 L, etc.

In certain embodiments, the yield of CMV complex (such as pentamericcomplex) is at least about 0.01 g/L, at least about 0.02 g/L, at leastabout 0.03 g/L, at least about 0.05 g/L, at least about 0.06 g/L, atleast about 0.07 g/L, at least about 0.08 g/L, at least about 0.09 g/L,at least about 0.1 g/L, at least about 0.15 g/L, at least about 0.20g/L, at least about 0.25 g/L, at least about 0.3 g/L, at least about0.35 g/L, at least about 0.4 g/L, at least about 0.45 g/L, at leastabout 0.5 g/L, at least about 0.55 g/L, at least about 0.6 g/L, at leastabout 0.65 g/L, at least about 0.7 g/L, at least about 0.75 g/L, atleast about 0.8 g/L, at least about 0.85 g/L, at least about 0.9 g/L, atleast about 0.95 g/L, or at least about 1.0 g/L.

Also provided herein is a process of producing cytomegalovirus (CMV) gLprotein, or a fragment thereof, or a complex comprising said gL proteinor fragment, comprising: (i) culturing the host cell described hereinunder a suitable condition, thereby expressing said gL protein, orfragment thereof; and (ii) harvesting said gL protein, or fragmentthereof, or the complex comprising said gL protein or fragment, from theculture.

In certain embodiments, the gL protein (or fragment thereof), or complexcomprising a complex comprising said gL protein or fragment describedherein is purified. The gL protein (or fragment thereof) can be purifiedusing any suitable methods, such as HPLC, various types ofchromatography (such as hydrophobic interaction, ion exchange, affinity,chelating, and size exclusion), electrophoresis, density gradientcentrifugation, solvent extraction, or the like.

For example, ion exchange may be used to purify the gL protein (orfragment thereof), or complex comprising a complex comprising said gLprotein or fragment. Examples of materials useful in the ion exchangechromatography include DEAE-cellulose, QAE-cellulose, DEAE-cephalose,QAE-cephalose, DEAE-Toyopearl, QAE-Toyopearl, Mono Q, Mono S, Qsepharose, SP sepharose, etc. In one exemplary embodiment, the methoduses a Mono S column. In another exemplary embodiment, the method uses aMono Q column.

Alternatively or in addition, affinity-based purification may be used.Examples of affinity-purification tags include, e.g., His tag (binds tometal ion), an antibody (binds to protein A or protein G),maltose-binding protein (MBP) (binds to amylose),glutathione-S-transferase (GST) (binds to glutathione), FLAG tag(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (SEQ ID NO: 8) (binds to an anti-flagantibody), Strep tag (binds to streptavidin or a derivative thereof).

One exemplary embodiment is Strep tag (or streptavidin affinity tag), atag that binds to streptavidin or a derivative thereof, such asStrep-Tactin. Strep tag comprises a peptide of nine amino acids:Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 9), or eight amino acids(also called strep-tag II): Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:10). Elution of a protein attached to a strep-tag from the column can beperformed using biotin or a derivative or homologue thereof, such asdesthio-biotin.

The affinity-purification tag may be attached by any suitable means, andmay be attached directly or indirectly. For example, the tag may becovalently attached at the N-terminus of the polypeptide sequence, or atthe C-terminus of the polypeptide sequence. This can be achieved byrecombinant expression of a fusion protein comprising the polypeptideand the tag, or by standard conjugation techniques that links thepolypeptide to the tag. The tag may be attached to the side chainfunctional group of an amino acid residue of the polypeptide usingstandard conjugation techniques. Alternatively, the tag may be attachednon-covalently.

Attachment of the tag may be direct, or indirect (through a linker).Suitable linkers are known to those skilled in the art and include,e.g., straight or branched-chain carbon linkers, heterocyclic carbonlinkers, carbohydrate linkers and polypeptide linkers.

In a certain embodiment, cleavable linkers may be used to attach themolecule of interest to the tag. This allows for the tag to be separatedfrom the purified complex, for example by the addition of an agentcapable of cleaving the linker. A number of different cleavable linkersare known to those of skill in the art. Such linkers may be cleaved forexample, by irradiation of a photolabile bond or acid-catalyzedhydrolysis. There are also polypeptide linkers which incorporate aprotease recognition site and which can be cleaved by the addition of asuitable protease enzyme.

When a complex comprising the gL protein (or fragment thereof) ispurified, the tag can be attached to other constituent(s) of thecomplex. For example, when purifying CMV pentameric complex, a tag maybe attached to pUL128, pUL130, or pUL131.

5. Pharmaceutical Compositions and Administration

The invention also provides pharmaceutical compositions comprising theCMV proteins, complexes, and nucleic acids described herein. Theinvention also provides pharmaceutical compositions comprising nucleicacid encoding CMV proteins, complexes, and nucleic acids describedherein.

The CMV proteins, complexes, and nucleic acids described herein can beincorporated into an immunogenic composition, or a vaccine composition.Such compositions can be used to raise antibodies in a mammal (e.g. ahuman).

The invention provides pharmaceutical compositions comprising the CMVproteins, complexes, and nucleic acids described herein, and processesfor making a pharmaceutical composition involving combining the CMVproteins, complexes, and nucleic acids described herein with apharmaceutically acceptable carrier. The pharmaceutical compositions ofthe invention typically include a pharmaceutically acceptable carrier,and a thorough discussion of such carriers is available in Remington:The Science and Practice of Pharmacy.

The pH of the composition is usually between about 4.5 to about 11, suchas between about 5 to about 11, between about 5.5 to about 11, betweenabout 6 to about 11, between about 5 to about 10.5, between about 5.5 toabout 10.5, between about 6 to about 10.5, between about 5 to about 10,between about 5.5 to about 10, between about 6 to about 10, betweenabout 5 to about 9.5, between about 5.5 to about 9.5, between about 6 toabout 9.5, between about 5 to about 9, between about 5.5 to about 9,between about 6 to about 9, between about 5 to about 8.5, between about5.5 to about 8.5, between about 6 to about 8.5, between about 5 to about8, between about 5.5 to about 8, between about 6 to about 8, about 4.5,about 5, about 6.5, about 6, about 6.5, about 7, about 7.5, about 8,about 8.5, about 9, about 9.5, about 10, about 10.5, about 11 , etc.Stable pH may be maintained by the use of a buffer e.g. a Tris buffer, acitrate buffer, a phosphate buffer, or a histidine buffer. Thus acomposition will generally include a buffer.

A composition may be sterile and/or pyrogen free. Compositions may beisotonic with respect to humans.

A composition comprises an immunologically effective amount of itsantigen(s). An “immunologically effective amount” is an amount which,when administered to a subject, is effective for eliciting an antibodyresponse against the antigen. This amount can vary depending upon thehealth and physical condition of the individual to be treated, theirage, the capacity of the individual's immune system to synthesizeantibodies, the degree of protection desired, the formulation of thevaccine, the treating doctor's assessment of the medical situation, andother relevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine trials.The antigen content of compositions of the invention will generally beexpressed in terms of the mass of protein per dose. A dose of 10-500 μg(e.g. 50 μg) per antigen can be useful.

Immunogenic compositions may include an immunological adjuvant.Exemplary adjuvants include mineral-containing compositions; oilemulsions; saponin formulations; virosomes and virus-like particles;bacterial or microbial derivatives; bioadhesives and mucoadhesives;liposomes; polyoxyethylene ether and polyoxyethylene ester formulations;polyphosphazene (pcpp); muramyl peptides; imidazoquinolone compounds;thiosemicarbazone compounds; tryptanthrin compounds; humanimmunomodulators; lipopeptides; benzonaphthyridines; microparticles;immunostimulatory polynucleotide (such as RNA or DNA; e.g.,CpG-containing oligonucleotides).

For example, the composition may include an aluminum salt adjuvant, anoil in water emulsion (e.g. an oil-in-water emulsion comprisingsqualene, such as MF59 or AS03), a TLR7 agonist (such asimidazoquinoline or imiquimod), or a combination thereof. Suitablealuminum salts include hydroxides (e.g. oxyhydroxides), phosphates (e.g.hydroxyphosphates, orthophosphates), (e.g. see chapters 8 & 9 of VaccineDesign (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum), ormixtures thereof. The salts can take any suitable form (e.g. gel,crystalline, amorphous, etc.), with adsorption of antigen to the saltbeing an example. The concentration of Al⁺³ in a composition foradministration to a patient may be less than 5 mg/ml e.g. <4 mg/ml, <3mg/ml, <2 mg/ml, <1 mg/ml, etc. A preferred range is between 0.3 and 1mg/ml. A maximum of 0.85 mg/dose is preferred. Aluminum hydroxide andaluminum phosphate adjuvants are suitable for use with the invention.

One suitable immunological adjuvant comprises a compound of Formula (I)as defined in WO2011/027222, or a pharmaceutically acceptable saltthereof, adsorbed to an aluminum salt. Many further adjuvants can beused, including any of those disclosed in Powell & Newman (1995).

Compositions may include an antimicrobial, particularly when packaged inmultiple dose format. Antimicrobials such as thimerosal and 2phenoxyethanol are commonly found in vaccines, but sometimes it may bedesirable to use either a mercury-free preservative or no preservativeat all.

Compositions may comprise detergent e.g. a polysorbate, such aspolysorbate 80. Detergents are generally present at low levels e.g.<0.01%.

Compositions may include sodium salts (e.g. sodium chloride) to givetonicity. A concentration of 10±2 mg/ml NaCl is typical, e.g., about 9mg/ml.

In another aspect, the invention provides a method of inducing an immuneresponse against cytomegalovirus (CMV), comprising administering to asubject in need thereof an immunologically effective amount of theimmunogenic composition describe herein, which comprises the proteins,DNA molecules, RNA molecules (e.g., self-replicating RNA molecules), orVRPs as described above.

In certain embodiments, the immune response comprises the production ofneutralizing antibodies against CMV. In certain embodiments, theneutralizing antibodies are complement-independent.

The immune response can comprise a humoral immune response, acell-mediated immune response, or both. In some embodiments an immuneresponse is induced against each delivered CMV protein. A cell-mediatedimmune response can comprise a Helper T-cell (Th) response, a CD8+cytotoxic T-cell (CTL) response, or both. In some embodiments the immuneresponse comprises a humoral immune response, and the antibodies areneutralizing antibodies. Neutralizing antibodies block viral infectionof cells. CMV infects epithelial cells and also fibroblast cells. Insome embodiments the immune response reduces or prevents infection ofboth cell types. Neutralizing antibody responses can becomplement-dependent or complement-independent. In some embodiments theneutralizing antibody response is complement-independent. In someembodiments the neutralizing antibody response is cross-neutralizing;i.e., an antibody generated against an administered compositionneutralizes a CMV virus of a strain other than the strain used in thecomposition.

A useful measure of antibody potency in the art is “50% neutralizationtiter.” To determine 50% neutralization titer, serum from immunizedanimals is diluted to assess how dilute serum can be yet retain theability to block entry of 50% of viruses into cells. For example, atiter of 700 means that serum retained the ability to neutralize 50% ofvirus after being diluted 700-fold. Thus, higher titers indicate morepotent neutralizing antibody responses. In some embodiments, this titeris in a range having a lower limit of about 200, about 400, about 600,about 800, about 1000, about 1500, about 2000, about 2500, about 3000,about 3500, about 4000, about 4500, about 5000, about 5500, about 6000,about 6500, or about 7000. The 50% neutralization titer range can havean upper limit of about 400, about 600, about 800, about 1000, about1500, about 2000, about 2500, about 3000, about 3500, about 4000, about4500, about 5000, about 5500, about 6000, about 6500, about 7000, about8000, about 9000, about 10000, about 11000, about 12000, about 13000,about 14000, about 15000, about 16000, about 17000, about 18000, about19000, about 20000, about 21000, about 22000, about 23000, about 24000,about 25000, about 26000, about 27000, about 28000, about 29000, orabout 30000. For example, the 50% neutralization titer can be about 3000to about 25000. “About” means plus or minus 10% of the recited value.

Compositions of the invention will generally be administered directly toa subject. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by any other suitableroute. For example, intramuscular administration may be used e.g. to thethigh or the upper arm. Injection may be via a needle (e.g. a hypodermicneedle), but needle-free injection may alternatively be used. A typicalintramuscular dosage volume is about 0.5 ml.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunization schedule and/or ina booster immunization schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes, e.g., a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

The subject may be an animal, preferably a vertebrate, more preferably amammal. Exemplary subject includes, e.g., a human, a cow, a pig, achicken, a cat or a dog, as the pathogens covered herein may beproblematic across a wide range of species. Where the vaccine is forprophylactic use, the human is preferably a child (e.g., a toddler orinfant), a teenager, or an adult; where the vaccine is for therapeuticuse, the human is preferably a teenager or an adult. A vaccine intendedfor children may also be administered to adults, e.g., to assess safety,dosage, immunogenicity, etc.

Vaccines of the invention may be prophylactic (i.e. to prevent disease)or therapeutic (i.e. to reduce or eliminate the symptoms of a disease).The term prophylactic may be considered as reducing the severity of orpreventing the onset of a particular condition. For the avoidance ofdoubt, the term prophylactic vaccine may also refer to vaccines thatameliorate the effects of a future infection, for example by reducingthe severity or duration of such an infection.

Isolated and/or purified CMV proteins, complexes, and nucleic acidsdescribed herein can be administered alone or as either prime or boostin mixed-modality regimes, such as a RNA prime followed by a proteinboost. Benefits of the RNA prime protein boost strategy, as compared toa protein prime protein boost strategy, include, for example, increasedantibody titers, a more balanced IgG1:IgG2a subtype profile, inductionof TH1-type CD4+ T cell-mediated immune response that was similar tothat of viral particles, and reduced production of non-neutralizingantibodies. The RNA prime can increase the immunogenicity ofcompositions regardless of whether they contain or do not contain anadjuvant.

In the RNA prime-protein boost strategy, the RNA and the protein aredirected to the same target antigen. Examples of suitable modes ofdelivering RNAs include virus-like replicon particles (VRPs), alphavirusRNA, replicons encapsulated in lipid nanoparticles (LNPs) or formulatedRNAs, such as replicons formulated with cationic nanoemulsions (CNEs).Suitable cationic oil-in-water nanoemulsions are disclosed inWO2012/006380 e.g. comprising an oil core (e.g. comprising squalene) anda cationic lipid (e.g. DOTAP, DMTAP, DSTAP, DC-cholesterol, etc.).

WO2012/051211 discloses that antibodies to the pentameric complex areproduced in mice that have been immunized with VRPs and formulated RNAs(CNEs and LNPs) that encode the protein constituents of the pentamericcomplex. These antibodies have been found to be capable of neutralizingCMV infection in epithelial cells. The RNA prime-protein boost regimenmay involve first (e.g. at weeks 0-8) performing one or more primingimmunization(s) with RNA (which could be delivered as VRPs, LNPs, CNEs,etc.) that encodes one or more of the protein components of a CMVprotein complex of the invention and then perform one or more boostingimmunization(s) later (e.g. at weeks 24-58) with: an isolated CMVprotein complex of the invention, optionally formulated with an adjuvantor a purified CMV protein complex of the invention, optionallyformulated with an adjuvant.

In some embodiments, the RNA molecule is encapsulated in, bound to oradsorbed on a cationic lipid, a liposome, a cochleate, a virosome, animmune-stimulating complex, a microparticle, a microsphere, ananosphere, a unilamellar vesicle, a multilamellar vesicle, anoil-in-water emulsion, a water-in-oil emulsion, an emulsome, apolycationic peptide, a cationic nanoemulsion, or combinations thereof.

Also provided herein are kits for administration of nucleic acid (e.g.,RNA), purified proteins, and purified complexes described herein, andinstructions for use. The invention also provides a delivery devicepre-filled with a composition or a vaccine disclosed herein.

The pharmaceutical compositions described herein can be administered incombination with one or more additional therapeutic agents. Theadditional therapeutic agents may include, but are not limited toantibiotics or antibacterial agents, antiemetic agents, antifungalagents, anti-inflammatory agents, antiviral agents, immunomodulatoryagents, cytokines, antidepressants, hormones, alkylating agents,antimetabolites, antitumor antibiotics, antimitotic agents,topoisomerase inhibitors, cytostatic agents, anti-invasion agents,antiangiogenic agents, inhibitors of growth factor function inhibitorsof viral replication, viral enzyme inhibitors, anticancer agents,α-interferons, β-interferons, ribavirin, hormones, and other toll-likereceptor modulators, immunoglobulins (Igs), and antibodies modulating Igfunction (such as anti-IgE (omalizumab)).

In certain embodiments, the compositions disclosed herein may be used asa medicament, e.g., for use in inducing or enhancing an immune responsein a subject in need thereof, such as a mammal.

In certain embodiments, the compositions disclosed herein may be used inthe manufacture of a medicament for inducing or enhancing an immuneresponse in a subject in need thereof, such as a mammal.

One way of checking the efficacy of therapeutic treatment involvesmonitoring pathogen infection after administration of the compositionsor vaccines disclosed herein. Another way of checking the efficacy ofprophylactic treatment involves monitoring immune responses,systemically (such as monitoring the level of IgG1 and IgG2a production)and/or mucosally (such as monitoring the level of IgA production),against the antigen. Typically, antigen-specific serum antibodyresponses are determined post-immunization but pre-challenge whereasantigen-specific mucosal antibody responses are determinedpost-immunization and post-challenge.

This invention is further illustrated by the following examples whichshould not be construed as limiting.

EXAMPLES Example 1 Materials and Methods

Sequence and structure analysis. gL sequences of CMV, VZV and HSV1 andHSV2 were aligned using CLUSTALW (Hyper Text Transfer Protocol Secure(https)://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clustalw.html)and manually adjusted to align residues contributed to conservedp-strands in VZV and HSV2.

Expression of penta and gH/gL complex. Wild type (WT) pentameric complex(“penta”) or penta with gL mutations (“LSG” and “IDG” mutant) wereexpressed using a two vector system with gH and gL in one vector, andthree ULs in the other. The sequence of IRES (internal ribosome entrysite) separates different genes in each vector. gH has a C-terminal 6×His tag (SEQ ID NO: 11), and UL130 has a cleavable C-terminal strep-tag.DNA of the two vectors with 1 mg of total DNA for every liter of culturewere transfected into Expi293 cells using Expifectamine transfection kit(Life Technologies) following the manufacture's protocol. Cells weregrown to ˜2.5×10⁶ cells/mL on the day of transfection withviability >97% in shaker flasks. The transfected cells were grown forthree days to ˜8×10⁶ cells/mL with viability ˜60% in shaker incubatoroperated at 37° C., 150 rpm and 8% CO₂. Supernatants of the expressionmedia were harvested by centrifugation at 4200 rpm for 30 minutes.

WT gH/gL or gH/gL with gL mutations were expressed using the vectorcontaining both gH and gL in the same way as described above.

N-terminal sequencing. N-terminal sequencing was used to identifyunknown bands visible by SDS pages and western blots (WBs) of affinitypurified WT penta. Penta on a SDS page was transferred to an ethanolactivated PVDF membrane, which was stained by 0.02% Coomassie Brilliantblue in 40% methanol, then washed in distilled water several timesbefore air dried completely. Bands of interest were cut out and shippedto Tufts University Protein Core Facility for sequencing.

Purification and western blot analysis. Harvested supernatant wasconcentrated and buffer exchanged into affinity column binding buffer(50 mM Hepes pH7.0, 150 mM NaCl, and 1 mM EDTA) using a KrosFlo ResearchII TFF system and hollow Fiber Cartridge (Spectrumlabs). Concentratedsupernatant was loaded to StrepTrap HP cartridge (GE Life Sciences), andeluted with elution buffer (50 mM Hepes pH 7.0, 150 mM NaCl, 2.5 mMdesthiobiotin, and 1 mM EDTA). Peak fractions from the eluate wereanalyzed by SDS-PAGE and western blotting using antibodies againsteither gL or the His-tag placed at the C-terminus of gH.

Immunization studies in mice. Ten mice per group were immunized withpurified WT or mutant pentameric complex gH/gL/pUL128/pUL130/pUL131adjuvanted with MF59 at the three different doses 0.03 μg, 0.1 μg and 1μg with three injections at three week intervals. Serum samples wereheat-inactivated at 56° C. for 30 min, serially diluted in two-foldsteps (two replicates per dilution), mixed with an equal volume of HCMVvirus diluted to a target concentration of 200-250 infectedcells/counting field in media±10% guinea pig complement (Cedarlane Labs,Burlington, N.C., USA), and incubated for 2 h at 37° C./5% CO₂. Theseserum/virus samples were added to ARPE-19 cells or MRC-5 cells preparedin 96-well half-area cell culture plates (Corning Inc., Corning, N.Y.,USA). The infected monolayers were incubated for 48 hours (±8 h) at 37°C./5% CO2, fixed with 10% buffered formalin (EMD Chemicals Inc.,Gibbstown, N.J., USA) for one hour and washed three times with washbuffer (PBS/0.05% Tween-20), blocked with PBS/2.5% fetal bovine serum,0.5% saponin, 0.1% sodium azide for one hour at room temperature. Theplates were washed three times, taped dry and incubated in a 25° C.humid incubator for one hour. The plates were then incubated for onehour at room temperature with anti-HCMV IE1 antibody derived fromhybridoma L14 (diluted in saponin buffer). Plates were washed threetimes and incubated for one hour with anti-mouse IgG conjugated withAlexaFluor 488 (diluted in saponin buffer), and then washed three timeswith PBS/0.05% Tween-20. The fluorescent cells were counted using anImmunospot S5 UV Analyzer (Cellular Technology Limited, Shaker Heights,Ohio, USA), and the 50% neutralization titer, defined as the reciprocalof the serum dilution yielding 50% reduction in the infected cell count(relative to infected cell count in diluent plus virus control wells),was calculated by linear regression interpolation between the twodilutions with wells yielding average infected cell counts above andbelow the 50% value.

Example 2 Results

1. gL Clipping Occurs Next to a Conserved β-Strand

N-terminal sequencing determined that a band in identified by westernblot using an antibody to gL begins with gL residue 97. Thus thegH/gL/pUL128/pUL130/pUL131 pentameric complex expressed in mammaliancells contains a population of gL proteins clipped between gL residuesAsn97 and Ser98. Structure based sequence alignment further disclosedthat the clipping site is in a loop region next to a β-strand conservedin both VZV and HSV-2 gH/gL structures (FIGS. 1A-1B).

Various mutations introduced into the gL sequence in the vicinity ofthis clipping site resulted in a reduction in the amount of gL clipping.Addition mutations inserted between two and five residues with a mix ofpolar and nonpolar residues into the cleavage site. Deletion mutationsdeleted from one to three residues around the cleavage site.Substitution mutations changed Ala96 to hydrophobic residues or residueswith large side-chains; changed Asn97 to polar residues with eithersmaller or larger side-chains, or to nonpolar residues; or changed Ser98to residues with small side-chains having either a polar or nonpolarcharacter.

2. Comparison of Various Mutant gH/gL with Wild Type gH/gL

FIG. 2A shows various mutant gL proteins that were tested. gH/gLcomplexes containing various gL mutations were expressed in Expi293cells and compared to WT gH/gL. Mutations at the Protease RecognitionSite reduced gL clipping in the expressed gH/gL complexes. For example,an anti-gL western blot of WT raw supernatant showed a clearly visibleband of gL fragment with residue 98 at its N-terminus, as determined byN-terminal sequencing. In contrast, a similar band was not detected inthe “LSG” mutant and was either not detected or significantly reduced inthe “delta Asn97” and “SST” mutants (FIGS. 2A-2C).

The three residue variants introduced in the vicinity of the clippingsite reduced clipping to a greater extent than a single residue variantin the vicinity of the clipping site. The “LSG” mutant reduced theintensity of gL clipping band most significantly in anti-His westernblot. In addition, removing the 17-residue insertion enhanced theintensity of the gL clipping band observed by western blot, gH/gL(N),suggesting that this insertion may protect the cleavage site (FIG. 2C).

3. Comparison of “LSG” and “IDG” Mutant Penta with Wild Type Pentamer

To analyze whether the “LSG and “IDG” mutants also eliminated or reducedgL clipping in the gH/gL/pUL128/pUL130/pUL131 pentamer, affinitypurified WT and mutant pentamer were analyzed by anti-His and anti-gLwestern blots. In the anti-His western blot of WT pentamer, there is apronounced band with smaller molecular weight than the full-lengthgH/gL, consistent with a complex of gH and the N-terminal region of gLafter clipping. Note that this N-terminal fragment of gL is notrecognized by the anti-gL antibody. In the anti-gL western blot, theC-terminal region of gL, beginning with residue 98 as determined byN-terminal sequencing, forms a complex with UL128 in a non-reducedsample. The same C-terminal fragment of gL by itself was observed in areduced sample. In comparison, those bands resulting from gL clippingwere not detected in either the “LSG” or “IDG” mutant pentamers (FIGS. 3and 4). Both “LSG” and “IDG” mutants produced pentamer that behavedsimilarly to WT pentamer complex. Therefore those mutations do notaffect the assembly of the gH/gL/pUL128/pUL130/pUL131 pentamer complex,but eliminated the proteolytic clipping of the gL protein (FIG. 5A).

Immunogenicity analysis showed that the LSG and IDG mutants did notcompromise the immunogenicity of the gH/gL/pUL128/pUL130/pUL131pentameric complex (FIG. 5B).

The fragment of gL that resulted from clipping was detected during theexpression of the gH/gL, gH/gL/gO (data not shown) andgH/gL/pUL128/pUL130/pUL131 complexes. The clipping sites in these threecomplexes are identical, between gL residues 97 and 98. Therefore,mutations that prevent gL clipping during the expression of gH/gL alsoprevent gL clipping during the expression of the gH/gL/gO andgH/gL/pUL128/pUL130/pUL131 pentamer complexes.

With as few as three residue substitutions or a single deletion, gLclipping can, respectively, be eliminated or significantly reduced. Thelocation of these mutations is not expected to affect the conservedsecondary structure in their vicinity. This allows the production ofhomogenous gH/gL/pUL128/pUL130/pUL131 pentamer with its threedimensional structure, and antigenicity/immunogenicity largelyunaffected. We conclude that the strategy of mutating the sequence inthe vicinity of the clipping site with homologous sequence provedeffective.

The various features and embodiments of the present invention, referredto in individual sections above apply, as appropriate, to othersections, mutatis mutandis. Consequently, features specified in onesection may be combined with features specified in other sections, asappropriate.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications andpatents cited in this disclosure are incorporated by reference in theirentirety. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. The term “comprising”encompasses “including” as well as “consisting” e.g. a composition“comprising” X may consist exclusively of X or may include somethingadditional, e.g. X+Y.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do no materiallyalter the basic and novel characteristics of the claimed composition,method or structure. The term “consisting of” is generally taken to meanthat the invention as claimed is limited to those elements specificallyrecited in the claim (and may include their equivalents, insofar as thedoctrine of equivalents is applicable).

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following embodiments.

-   1. A recombinant cytomegalovirus (CMV) gL protein, or a    complex-forming fragment thereof, wherein said gL protein or    fragment comprises a mutation within Protease Recognition Site,    wherein said mutation reduces protease cleavage at said Protease    Recognition Site as compared to a control.-   2. The gL protein or fragment of embodiment 1, wherein said mutation    comprises addition, deletion, substitution, or a combination    thereof, of an amino acid residue.-   3. The gL protein or fragment of embodiment 1 or 2, wherein three or    more residues of said Protease Recognition Site form a β-strand, and    said mutation maintains the β-strand conformation.-   4. The gL protein or fragment of any one of embodiments 1-3, wherein    said mutation results in no more than 20% (molar percentage) of gL    cleaved at said Protease Recognition Site when recombinantly    expressed in a mammalian host cell.-   5. The gL protein or fragment of any one of embodiments 1-4, wherein    said mutation comprises addition of one or more amino acid residues.-   6. The gL protein or fragment of any one of embodiments 1-5, wherein    said mutation comprises addition of two to five amino acid residues.-   7. The gL protein or fragment of embodiment 6, wherein said two to    five amino acid residues comprise both polar residue(s) and    non-polar residue(s).-   8. The gL protein or fragment of any one of embodiments 1-7, wherein    said mutation comprises addition of one or more residues between    residues N97 and S98.-   9. The gL protein or fragment of any one of embodiments 1-8, wherein    said mutation comprises addition of F, Q, FQ or QF between residues    N97 and S98.-   10. The gL protein or fragment of any one of embodiments 1-9,    wherein said mutation comprises deletion of one or more amino acid    residues.-   11. The gL protein or fragment of any one of embodiments 1-10,    wherein said mutation comprises deletion of one to three amino acid    residues.-   12. The gL protein or fragment of any one of embodiments 1-11,    wherein said mutation comprises deletion of a residue selected from    the group consisting of: V91, T92, P93, E94, A95, A96, N97, S98,    V99, L100, L101, D102 and a combination thereof.-   13. The gL protein or fragment of any one of embodiments 1-12,    wherein said mutation comprises deletion of a residue selected from    the group consisting of: A95, A96, N97, and a combination thereof.-   14. The gL protein or fragment of any one of embodiments 1-13,    wherein said mutation comprises deleting N97.-   15. The gL protein or fragment of any one of embodiments 1-14,    wherein said mutation comprises substituting a residue with a    corresponding residue from a gL protein of another herpes virus.-   16. The gL protein or fragment of embodiment 15, wherein said gL    protein from another herpes virus is a gL protein from HSV1, HSV2,    VZV, EBV, PrV, or bovine herpesvirus 5.-   17. The gL protein or fragment of any one of embodiments 1-16,    wherein said mutation comprises substituting g A96 with a non-polar    residue or with a residue that comprises a large side chain.-   18. The gL protein or fragment of any one of embodiments 1-17,    wherein said mutation comprises substituting A96 with I, L, V or S.-   19. The gL protein or fragment of any one of embodiments 1-18,    wherein said mutation comprises substituting A95 with R, L, E or N.-   20. The gL protein or fragment of any one of embodiments 1-19,    wherein said mutation comprises substituting E94 with A or L.-   21. The gL protein or fragment of any one of embodiments 1-20,    wherein said mutation comprises substituting N97 with a polar    residue or a non-polar residue.-   22. The gL protein or fragment of embodiment 21, wherein said polar    residue comprises a small side chain.-   23. The gL protein or fragment of embodiment 21, wherein said polar    residue comprises a large side chain.-   24. The gL protein or fragment of any one of embodiments 1-23,    wherein said mutation comprises substituting N97 with S, D, E, A, T    or Y.-   25. The gL protein or fragment of any one of embodiments 1-24,    wherein said mutation comprises substituting N97 with S or D.-   26. The gL protein or fragment of any one of embodiments 1-25,    wherein said mutation comprises substituting S98 with an amino acid    residue with a small side chain.-   27. The gL protein or fragment of any one of embodiments 1-26,    wherein said mutation comprises substituting S98 with G, T, V, or I.-   28. The gL protein or fragment of any one of embodiments 1-27,    wherein said mutation comprises substituting S98 with G, or T.-   29. The gL protein or fragment of any one of embodiments 1-28,    wherein said mutation comprises substituting V99 with I.-   30. The gL protein or fragment of any one of embodiments 1-29,    wherein said mutation comprises substituting L100 with an amino acid    residue with F or V.-   31. The gL protein or fragment of any one of embodiments 1-30,    wherein said mutation comprises substituting L101 with an amino acid    residue with V or I.-   32. The gL protein or fragment of any one of embodiments 1-31,    wherein said gL protein or fragment comprises an Insert Region at    the N-terminus of the Protease Recognition Site.-   33. The gL protein or fragment of any one of embodiments 1-32,    wherein said mutation comprises introducing a non-naturally    occurring amino acid residue.-   34. The gL protein or fragment of any one of embodiments 1-33,    wherein said mutation comprises introducing an amino acid residue    comprising a bulky side chain.-   35. A CMV complex comprising the recombinant gL protein or fragment    of any one of embodiments 1-34.-   36. The complex of embodiment 35, comprising a CMV protein selected    from the group consisting of gH, gL, pUL128, pUL130, pUL131, gO, a    complex-forming fragment thereof, and a combination thereof.-   37. The complex of embodiment 35 or 36, wherein said complex is a    pentameric complex comprising: gH or a pentamer-forming fragment    thereof, gL or a pentamer-forming fragment thereof, pUL128 or a    pentamer-forming fragment thereof, pUL130 or a pentamer-forming    fragment thereof, and pUL131 or a pentamer-forming fragment thereof.-   38. The complex of embodiment 35 or 36, wherein said complex is a    gH/gL complex comprising: gH or a complex-forming fragment thereof,    and gL or a complex-forming fragment thereof.-   39. The complex of embodiment 35 or 36, wherein said complex is a    trimeric complex comprising: gH or a complex-forming fragment    thereof, gL or a complex-forming fragment thereof, and gO or a    complex-forming fragment thereof.-   40. An immunogenic composition comprising the recombinant CMV gL    protein or fragment of any of one of embodiments 1-34, or the    complex of any one of embodiment 35-39.-   41. The immunogenic composition of embodiment 40, further comprising    an adjuvant.-   42. The immunogenic composition of embodiment 41, wherein said    adjuvant comprises an aluminum salt, a TLR7 agonist, an oil-in-water    emulsion, or a combination thereof.-   43. The immunogenic composition of embodiment 42, wherein said    oil-in-water emulsion is MF59.-   44. An isolated nucleic acid comprising a polynucleotide sequence    encoding the recombinant CMV gL protein or fragment of any one of    embodiments 1-34.-   45. The isolated nucleic acid of embodiment 44, wherein said    isolated nucleic acid is an RNA, preferably a self-replicating RNA.-   46. The isolated nucleic acid of embodiment 45, wherein said    self-replicating RNA is an alphavirus replicon.-   47. An alphavirus replication particle (VRP) comprising the    alphavirus replicon of embodiment 46.-   48. An immunogenic composition comprising the nucleic acid of any    one of embodiments 44-46.-   49. An immunogenic composition comprising the VRP of embodiment 47.-   50. The immunogenic composition of embodiment 48 or 49, further    comprising an adjuvant.-   51. The immunogenic composition of embodiment 50, wherein said    adjuvant comprises an aluminum salt, a TLR7 agonist, an oil-in-water    emulsion (such as MF59), or a combination thereof.-   52. A host cell comprising the nucleic acid of any one of    embodiments 44-46.-   53. The host cell of embodiment 52, wherein said nucleic acid is a    DNA.-   54. The host cell of embodiment 53, wherein said host cell is a    mammalian cell.-   55. The host cell of embodiment 54, wherein said mammalian cell is a    CHO cell or HEK-293 cell.-   56. The host cell of any one of embodiments 53-55, wherein said DNA    encoding the CMV gL protein or fragment thereof is integrated into    the genomic DNA of said host cell.-   57. The host cell of any one of embodiments 52-56, wherein said host    cell comprises one or more polynucleotide sequences encoding CMV    pentameric complex, said pentameric complex comprising: gH or a    pentamer-forming fragment thereof, gL or a pentamer-forming fragment    thereof, pUL128 or a pentamer-forming fragment thereof, pUL130 or a    pentamer-forming fragment thereof, and pUL131 or a pentamer-forming    fragment thereof.-   58. The host cell of embodiment 57, wherein said one or more    polynucleotide sequences encoding CMV pentameric complex are    integrated into the genomic DNA of said host cell.-   59. The host cell of embodiment 57 or 58, wherein said cell, when    cultured under a suitable condition, expresses said CMV pentameric    complex.-   60. The host cell of embodiment 59, wherein said pentameric complex    is secreted.-   61. A cell culture comprising the host cell of any one of    embodiments 52-60, wherein said culture is at least 20 liter in    size.-   62. A cell culture comprising the host cell of any one of    embodiments 52-60, wherein said culture is at least 100 liter in    size.-   63. A cell culture comprising the host cell of any one of    embodiments 57-60, wherein the yield of said pentameric complex is    at least 0.05 g/L.-   64. A cell culture comprising the host cell of embodiment 63,    wherein the yield said pentameric complex is at least 0.1 g/L.-   65. A process of producing a recombinant cytomegalovirus (CMV) gL    protein, or a complex-forming fragment thereof, comprising:    -   (i) culturing the host cell of any one of embodiments 52-60        under a suitable condition, thereby expressing said gL protein,        or complex-forming fragment thereof; and    -   (ii) harvesting said gL protein, or complex-forming fragment        thereof, from the culture.-   66. A method of inducing an immune response against cytomegalovirus    (CMV), comprising administering to a subject in need thereof an    immunologically effective amount of the immunogenic composition of    any one of embodiments 40-43 and 48-51.-   67. The method of embodiment 66, wherein the immune response    comprises the production of neutralizing antibodies against CMV.-   68. The method of embodiment 67, wherein the neutralizing antibodies    are complement-independent.-   69. A method of inhibiting cytomegalovirus (CMV) entry into a cell,    comprising contacting the cell with the immunogenic composition of    any one of embodiments 40-43 and 48-51.-   70. The immunogenic composition of any one of embodiments 40-43 and    48-51 for use in inducing an immune response against cytomegalovirus    (CMV).-   71. Use of the immunogenic composition of any one of embodiments    40-43 and 48-51 for inducing an immune response against    cytomegalovirus (CMV).-   72. Use of the immunogenic composition of any one of embodiments    40-43 and 48-51 in the manufacture of a medicament for inducing an    immune response against cytomegalovirus (CMV).

Sequences SEQ ID NO: 1 (gL from HCMV strain Merlin = GI: 39842115)MCRRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRCLLGEVFEGDKYESWLRPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNVVVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR SEQ ID NO: 2 (gL from HCMV strain Towne =GI: 239909463) MCRRPDCGFSFSPGPVALLWCCLLLPIVSSATVSVAPTVAEKVPAECPELTRRCLLGEVFQGDKYESWLRPLVNVTRRDGPLSQLIRYRPVTPEAANSVLLDDAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLENVVVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR SEQ ID NO: 3 (gL from HCMV strain AD169 =GI: 2506510) MCRRPDCGFSFSPGPVVLLWCCLLLPIVSSVAVSVAPTAAEKVPAECPELTRRCLLGEVFQGDKYESWLRPLVNVTRRDGPLSQLIRYRPVTPEAANSVLLDDAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNVVVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDARSEQ ID NO: 4 (gL mature protein consisting ofamino acid residues 31-278 of SEQ ID NO: 1)AAVSVAPTAAEKVPAECPELTRRCLLGEVFEGDKYESWLRPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNVVVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDARSEQ ID NO: 5 (17 residue insert from gL from HCMV strain Merlin)GRDGPLSQLIRYRPVTP SEQ ID NO: 6 (gH from HCMV strain Towne = GI: 138314)MRPGLPSYLIVLAVCLLSHLLSSRYGAEAISEPLDKAFHLLLNTYGRPIRFLRENTTQCTYNSSLRNSTVVRENAISFNFFQSYNQYYVEHMPRCLFAGPLAEQFLNQVDLTETLERYQQRLNTYALVSKDLASYRSFSQQLKAQDSLGEQPTTVPPPIDLSIPHVWMPPQTTPHGWTESHTTSGLHRPHFNQTCILFDGHDLLFSTVTPCLHQGFYLIDELRYVKITLTEDFFVVTVSIDDDTPMLLIFGHLPRVLFKAPYQRDNFILRQTEKHELLVLVKKDQLNRHSYLKDPDFLDAALDFNYLDLSALLRNSFHRYAVDVLKSGRCQMLDRRTVEMAFAYALALFAAARQEEAGAQVSVPRALDRQAALLQIQEFMITCLSQTPPRTTLLLYPTAVDLAKRALWTPNQITDITSLVRLVYILSKQNQQHLIPQWALRQIADFALKLHKTHLASFLSAFARQELYLMGSLVHSMLVHTTERREIFIVETGLCSLAELSHFTQLLAHPHHEYLSDLYTPCSSSGRRDHSLERLTRLFPDATVPTTVPAALSILSTMQPSTLETFPDLFCLPLGESFSALTVSEHVSYVVTNQYLIKGISYPVSTTVVGQSLIITQTDSQTKCELTRNMHTTHSITAALNISLENCAFCQSALLEYDDTQGVINIMYMHDSDDVLFALDPYNEVVVSSPRTHYLMLLKNGTVLEVTDVVVDATDSRLLMMSVYALSAIIGIYLLYRMLKTCSEQ ID NO: 7 (6 residue insert from gL from HCMV strain Merlin) NSVLLDSEQ ID NO: 8 (FLAG Tag) DYKDDDDK SEQ ID NO: 9 (Strep Tag) AWRHPQFGGSEQ ID NO: 10 (Strep Tag II) WSHPQFEK SEQ ID NO: 11 (His Tag) HHHHHH

1-15. (canceled)
 16. A process of producing a recombinant HumanCytomegalovirus (HCMV) gL protein, or a complex-forming fragmentthereof, comprising: (i) culturing an isolated host cell under asuitable condition, wherein said isolated host cell comprises a nucleicacid that encodes a recombinant HCMV gL protein, or a complex-formingfragment thereof, having a substitution mutation at a residuecorresponding to A96, N97, S98, or a combination thereof, numberedaccording to SEQ ID NO: 1, thereby expressing said gL protein, orcomplex-forming fragment thereof; and (ii) harvesting said gL protein,or complex-forming fragment thereof, from the culture.
 17. A process ofproducing a Human Cytomegalovirus (HCMV) complex that comprises arecombinant gL protein, or a complex-forming fragment thereof,comprising: (i) culturing an isolated host cell under a suitablecondition, wherein said isolated host cell comprises a nucleic acid thatencodes an HCMV complex comprising a recombinant HCMV gL protein, or acomplex-forming fragment thereof, having a mutation at residues 91-102numbered according to SEQ ID NO: 1, thereby expressing said HCMVcomplex; and (ii) harvesting said HCMV complex from the culture.
 18. Theprocess of claim 17, wherein said HCMV complex is a pentameric complexcomprising said recombinant HCMV gL protein, or a pentamer-formingfragment thereof, and: an HCMV gH protein or a pentamer-forming fragmentthereof, an HCMV pUL128 protein or a pentamer-forming fragment thereof,an HCMV pUL130 protein or a pentamer-forming fragment thereof, and anHCMV pUL131 protein or a pentamer-forming fragment thereof.
 19. Theprocess of claim 18, wherein said HCMV pentamer complex comprises a gHpentamer-forming fragment that lacks a transmembrane domain.
 20. Theprocess of claim 17, wherein the complex comprises an HCMV gL protein,or complex-forming fragment thereof, having a substitution mutation at aresidue corresponding to A96, N97, S98, or a combination thereof,numbered according to SEQ ID NO:
 1. 21. The process of claim 20, whereinsaid HCMV complex is a pentameric complex comprising said recombinantHCMV gL protein, or a pentamer-forming fragment thereof, and: an HCMV gHprotein or a pentamer-forming fragment thereof, an HCMV pUL128 proteinor a pentamer-forming fragment thereof, an HCMV pUL130 protein or apentamer-forming fragment thereof, and an HCMV pUL131 protein or apentamer-forming fragment thereof.
 22. The process of claim 21, whereinsaid HCMV pentamer complex comprises a gH pentamer-forming fragment thatlacks a transmembrane domain.